The broad goal of this project continues to be to acquire basic knowledge at the molecular and cellular level about the molecular architecture and function of the endothelial cell surface and its caveolae. Having discovered lung-specific endothelial proteins and specific antibodies to such proteins, we have provided key "proof of concept" that the vascular targeting strategy can indeed meet theoretical expectations in achieving tissue-specific drug delivery after intravenous injection. Having discovered key mechanisms mediating trafficking of caveolae and establishing that caveolae can not only express tissue-specific proteins but also mediate select transport across the blood vessel wall, we have proposed and validated a new strategy of targeting caveolae for achieving tissue-specific delivery and also overcoming endothelial and epithelial cell barriers to pharmaco-delivery in vivo. Here we will focus on finishing our mapping of the lung endothelial cell surface and its caveolae, validating new putative lung-specific targets discovered through our proteomic analysis, and investigating signal regulation of budding and internalization of caveolae as well as the intracellular trafficking pathway of caveolae-targeted probes In order to realize these goals, this research proposal has the following specific aims i) to continue our proteomic analysis of the luminal cell surface of microvascular endothelium in rat lung tissue, including the mapping of caveolae, ii) to examine the transport function of caveolae and the intracellular trafficking of caveolar cargo through the use of novel caveolae targeting antibodies and other probes, iii) to define regulatory pathways for ligand-induced caveolar budding with emphasis on the role of heterotrimeric G proteins, G protein-coupled receptors, caveolin, and tyrosine phosphorylation Through studying the function of caveolae in transport in microvascular endothelium, we have begun, and will continue, to create new strategies for achieving organ-specific drug and gene delivery in vivo which appears essential for improving clinical efficacy in the treatment of many diseases. [unreadable] [unreadable]